Flow cytometry is a technique in which a fluid-flow system organizes cells within a stream of fluid such that the cells pass in single-file through a detection zone. As the cells pass through the detection zone, they are illuminated by laser light, which scatters from each cell in a manner that depends on its structure. Most modern flow cytometry approaches also employ numerous fluorochrome-labeled antibodies that selectively bind with specific cellular features, such as cell-associated molecules, proteins or ligands. When excited by light at their respective excitation wavelengths, each fluorochrome emits a characteristic fluorescence signal, indicating the presence of that fluorochrome-specific feature. The scattered light and fluorescence signals are detected and analyzed to classify and/or count the cells according to a set of parameters of interest. In some cases, once classified, the cells are sorted into sub-populations based on their particular characteristics.
Flow cytometry enables simultaneous multi-parameter analysis of individual cells in a fluid stream, such as analysis of cell surfaces and intracellular molecules, characterization and definition of different cell types in mixed cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. Flow cytometers are used in many clinical and biological applications, such as the diagnosis of blood cancers, basic research, clinical practice, and clinical trials.
Historically, fluid-flow systems in conventional flow cytometers have been of a stream-in-air configuration, in which the fluid stream is forced through a nozzle system so the cells pass in single file through a detection zone in open air. Other prior-art flow cytometers employ a flow cell configuration, wherein a sheath fluid hydrodynamically focuses the sample fluid into the core of an open stream that traverses the detection zone. Unfortunately, in each case, such prior-art flow cytometers have some significant disadvantages: (1) they are quite expensive; (2) they have a large footprint; (3) they are not easily portable; and (4) they require extensive time, expertise, and expense to use and maintain. In addition, systems having an open-flow design are difficult to adapt for use with infectious disease or pathogenic microbiological samples because of the risk of exposure.
To mitigate some of these disadvantages, microfluidics-based flow cytometers have been developed in which the sample fluid passes through the detection zone in an enclosed flow channel. The adoption of microfluidics approaches also enables increased on-chip functionality, such as filtering, cell sorting, and overall flow control.
Microfluidics-based flow cytometers are disclosed, for example, in U.S. Patent Publication No. 2009/0051912, which describes a flow cytometer system that is smaller and more portable than an open-flow system. In operation, the fluid-flow system is held under a microscope objective, which functions as an external optics system that provides the light used to interrogate the cells and collect light scattered or emitted from the detection zone.
In fact, most conventional flow cytometers rely on external optics for illuminating the detection zone and/or detecting the scattered light signals. Unfortunately, this limits how small and portable a flow cytometer can be made. In addition, careful alignment between the fluid-flow system and the external optics is critical for realizing precise and accurate measurements, and this alignment must be maintained during use to ensure proper system operation. Further exacerbating these issues, the use of several fluorochromes usually gives rise to a need for multiple lasers at different excitation wavelengths to excite the pallet of fluorochromes employed. Still further, numerous wavelength-filtered detectors are required to effectively discriminate between the resultant fluorescence signals. As a result, the use of external optics can add significant cost to a flow cytometry system.
Integrating optical surface waveguides with microfluidics fluid-flow systems offers some promise for mitigating some of the disadvantages of external optics-based flow cytometers. Examples of a microfluidics-based system having integrated optical surface waveguides are disclosed in U.S. Pat. No. 7,764,374, in which both fluid-flow channels and SU-8-based optical surface waveguides are formed on the top surface of a substrate. One SU-8 surface waveguide emits light into an analysis zone of the fluid-flow channel, while a second SU-8 surface waveguide, located across the fluid-flow channel, collects light after it has passed through the analysis zone.
In similar fashion, U.S. Patent Publication No. 2013/0083315 discloses flow cytometer arrangements having a first flow channel that includes a detection zone, and a plurality of “surface waveguide channels” that are adjacent to the detection zone. The surface waveguide channels are filled with fluid that laterally guides light captured from the detection zone to other regions of the substrate.
Unfortunately, such prior-art systems suffer from several disadvantages. It is often necessary to couple several independent light signals into or out of a single region. SU-8-based surface waveguides and fluid-filled surface waveguides require significant chip real estate, however. As a result, forming more than few optical surface waveguides that access the same location can be challenging.
Further, flow cytometry performance is improved when the detection zone is illuminated with substantially uniform light. Prior-art, microfluidics-based flow cytometers, however, are limited to providing illumination from one side of the fluid channel. As a result, uniform illumination of the sample fluid is precluded and system sensitivity is degraded.